# US 10,119,150 B2 · Feeder-free Derivation of human-induced pluripotent stem cells with synthetic messenger RNA

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,119,150 B2
- Document ID: US10119150B2
- Status: Granted
- Kind: B2
- Application number: 14/292,317
- Filed: 2014-05-30
- Published: 2014-11-27
- Granted: 2018-11-06
- Earliest claimed: 2012-05-13
- Assignees: Allele Biotechnology & Pharmaceuticals, Inc.
- Applicants: Allele Biotechnology & Pharmaceuticals, Inc.
- Inventors: Jiwu Wang
- Web page: https://patentgrep.com/patent/US10119150B2

## CPC

- C12N 15/85: for animal cells
- C07K 14/4702: {Regulators; Modulating activity}
- C07K 14/475: Growth factors; Growth regulators
- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C07K 2319/01: containing a localisation/targetting motif
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2320/30: Special therapeutic applications
- C12N 2501/602: Sox-2
- C12N 2501/603: Oct-3/4
- C12N 2501/605: Nanog
- C12N 2501/608: Lin28
- C12N 2506/1307: from adult fibroblasts
- C12N 2510/00: Genetically modified cells
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The present disclosure relates generally to novel methods and compositions for using engineered reprogramming factor(s) for the creation of induced pluripotent stem cells (iPSCs) through a kinetically controlled process. Specifically, this disclosure relates to establishing combinations of reprogramming factors, including fusions between conventional reprogramming factors with transactivation domains, optimized for reprogramming various types of cells. More specifically, the exemplary methods disclosed herein can be used for creating induced pluripotent stem cells from various mammalian cell types, including human fibroblasts. Exemplary methods of feeder-free derivation of human induced pluripotent stem cells using synthetic messenger RNA are also disclosed.

## Claims

### Claim 1

A method for dedifferentiating or reprogramming feeder-free, xeno-free and integration-free mammalian somatic cells to produce feeder-free, xeno-free and integration-free induced pluripotent stem cells comprising:
a) seeding mammalian somatic cells in a volume of 0.5 ml to 1 ml of xeno-free culture medium on a feeder-free and xeno-free surface;
b) plating the mammalian somatic cells from step a) at a cell density of 15,000 cells/ml to 500,000 cells/ml;
c) transfecting the plated mammalian somatic cells from step b) with a composition comprising:
1) one ml or less of xeno-free culture medium:
2) an effective amount of a fusion product between a synthetic mRNA encoding OCT4 and an N-terminal-linked transactivation domain, and
3) synthetic mRNA reprogramming factors encoding SOX2 KLF4, NANOG, and optionally c-MYC or LIN28, wherein the synthetic mRNA reprogramming factors are provided at a concentration of up to about 800 ng/ml;
d) culturing said somatic cells with the composition from step c) under feeder-free and xeno-free culture conditions until the media is changed once daily; and
e) repeating step d) until the mammalian somatic cells are reprogrammed or de-differentiated to produce feeder-free, xeno-free and integration-free induced pluripotent stem cells.

### Claim 2 (depends on 1)

The method of claim 1, wherein the synthetic mRNAs are purified to remove any contaminating non-mRNA species from transcription.

### Claim 3 (depends on 1)

The method of claim 1, wherein the cells in step a) are seeded in a density gradient.

### Claim 4 (depends on 1)

The method of claim 1, wherein the mammalian somatic cells are non-human primate cells.

### Claim 5 (depends on 1)

The method of claim 1, wherein the mammalian somatic cells are cynomolgus monkey cells.

### Claim 6 (depends on 1)

The method of claim 1, wherein the mammalian somatic cells are rhesus monkey cells.

### Claim 7 (depends on 1)

The method of claim 1, wherein the mammalian somatic cells are baboon cells.

### Claim 8 (depends on 1)

The method of claim 1, wherein the mammalian somatic cells are canine cells.

### Claim 9 (depends on 1)

The method of claim 1, wherein the mammalian somatic cells are equine cells.

### Claim 10 (depends on 1)

The method of claim 1, wherein the mammalian somatic cells are bovine cells.

### Claim 11 (depends on 1)

The method of claim 1, wherein the mammalian somatic cells are swine cells.

### Claim 12 (depends on 1)

The method of claim 1, wherein the cells mammalian somatic are ovine cells.

### Claim 13 (depends on 1)

The method of claim 1, wherein OCT4 is fused to an N-terminal MyoD transactivation domain in tandem triplicates.

### Claim 14 (depends on 4)

The method for reprogramming mammalian somatic cells of claim 4, wherein
steps d) and e) of the method further comprises transfecting the cells with varying doses of about 50 ng/ml to 800 ng/ml of synthetic mRNA reprogramming factors during each medium change, wherein lower doses of the synthetic mRNA reprogramming factors are used at earlier time points than later time points.

### Claim 15 (depends on 4)

The method of claim 4, wherein in step b) of the method further comprises plating the cells on a feeder-free surface at a cell density of about 15,000 cells/ml; 25,000 cells/ml; 50,000 cells/ml; 75,000 cells/ml; 100,000 cells/ml; or 150,000 cells/ml in xeno-free culture medium and culturing the non-human primate cells under feeder-free, xeno-free and integration-free conditions; and;
wherein step c) of the method further comprises transfecting the cells with varying doses of about 50 ng/ml to about 800 ng/ml of synthetic mRNA reprogramming factors during each medium change, wherein lower doses of the synthetic mRNA reprogramming factors are used at earlier time points than later time points.

### Claim 16 (depends on 4)

The method for reprogramming non-human mammalian somatic cells of claim 4, wherein step b) of the method further comprises:
1) plating the cells at a density of about 15,000 cells/ml; 25,000 cells/ml; 50,000 cells/ml; 75,000 cells/ml; 100,000 cells/ml; or 150,000 cells/ml in xeno-free culture medium and culturing the non-human primate cells under feeder-free, xeno-free and integration-free conditions wherein the volume of each surface is between 0.5 ml to 1 ml of xeno-free culture medium; and
wherein step c) of the method further comprises transfecting the cells with varying doses of about 50 ng/ml to about 800 ng/ml of synthetic mRNA reprogramming factors during each medium change, wherein lower doses of the synthetic mRNA reprogramming factors are used at earlier time points than later time points.

### Claim 17 (depends on 1)

The method of claim 1, wherein the induced pluripotent stem cells are maintained in culture.

### Claim 18 (depends on 1)

The method of claim 1, further comprising differentiating the induced pluripotent stem cells.

### Claim 19 (depends on 2)

The method of claim 2, wherein the composition further comprises Rarg and LrH 1 transactivation domains.

### Claim 20 (depends on 1)

The method of claim 1, wherein the fusion product comprises a synthetic mRNA encoding OCT4 fused to a VP16 transactivation domain.